It has been proposed that the contraction cycle in heart muscle may be regulated by changes in intracellular cAMP levels. There is considerable evidence that cyclic nucleotides and calcium are both required for regulation of heart contractility. Recent studies have indicated that cAMP may indirectly affect the contractile system by modulating the regulatory function of calcium ions and that calcium may in turn regulate the levels of cAMP and cGMP by affecting the calcium-sensitive phosphodiesterase and adenylate cyclase activities in heart muscle. The calcium sensitive regulatory protein (CDR) plays a key role in this process since it mediates calcium stimulation of PDE and apparently interacts with several other proteins in heart muscle. A clearer understanding of the interdependence between cyclic nucleotides and calcium in heart requires three fundamental studies which define the major objectives of this proposal: Detection and isolation of CDR binding components in heart; quantitation of the binding between CDR and CDR binding components, particularly the calcium-sensitive PDE; and reexamination of the calcium sensitivity of heart adenylate cyclase. Binding components will be purified by CDR-sepharose affinity chromatography and conventional purification techniques. Iodinated CDR will be used to detect CDR binding components in heart muscle. Procedures for fluorescence labeling of CDR have been developed in our laboratory and the equilibria and kinetics for the calcium dependent binding of CDR to PDE will be investigated using fluorescence techniques. This study should provide valuable information concerning regulation of cyclic nucleotide levels in heart muscle by calcium.